SUPPLEMENTARY APPENDIXRed Cell Biology & its Disorders

RON kinase inhibition reduces renal endothelial injury in sickle cell dis-ease miceAlfia Khaibullina,1 Elena A. Adjei,1,2 Nowah Afangbedji,1 Andrey Ivanov,1 Namita Kumari,1 Luis E.F. Almeida,3 ZenaideM.N. Quezado,3 Sergei Nekhai1,4,5 and Marina Jerebtsova5

1Center for Sickle Cell Disease, College of Medicine, Howard University, Washington, DC; 2Departments of Genetics and Human Genet-ics, College of Medicine, Howard University, Washington, DC; 3Department of Perioperative Medicine, NIH Clinical Center, National In-stitutes of Health, Bethesda, MD; 4Department of Medicine, College of Medicine, Howard University, Washington, DC and 5Departmentof Microbiology, College of Medicine, Howard University, Washington, DC, USA

©2018 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2017.180992

Received: September 15, 2017.

Accepted: March 1, 2018.

Pre-published: March 8, 2018.

Correspondence: marina.jerebtsova@howard.edu

Supplemental Methods

Mice

The animal protocol was approved by the Institutional Animal Care and Use Committee at the

Children's National Health System. We examined kidneys from B6:129-Hbatm1(HBA)Tow

Hbbtm2(HBG1,HBB*)Tow /Hbbtm3(HBG1,HBB)Tow/J strain, here referred to as the Townes (SCD) mice (1,

2). Townes mice (homozygous HBSS, and controls, HBAA) do not express mouse hemoglobin.

Homozygous Townes (hα/hα::βS/βS, HBSS) mice carry normal human α- (hα) and sickle

hemoglobin beta (βS) genes and express over 90% of human sickle hemoglobin. Control Townes

(hα/hα::βA/βA) mice have hα and βA genes and express normal human hemoglobin breeding pairs

were obtained from the Jackson Laboratory (stock numbers 013071 Bar Harbor, ME) and bred in

our animal facility. Genotypes for all animals were confirmed as previously described. Kidneys

were collected from four-month old SCD and control mice (5 mice per group) and used for

microscopic evaluation.

Immunohistochemistry

Paraffin embedded tissues sections were used for immunostaining with rat anti-mouse F4/80

(Cat# MCA497, AbD Serotec), rabbit anti-vWF (Cat# IR52761-2, Dako), rat anti- mouse ICAM

(Cat# 116120, BioLegend), rat anti-mouse CD34 (Cat#CL8927AP, Cedarline), and mouse anti-

MSP1 (Cat# MAB735, R&D Systems). AEC (red staining) and DAB (brown staining) kits were

obtained from Vector Laboratory. PCNA kit was obtained from Invitrogen (Cat#93-1143).

Images were acquired by Olympus 1x51 microscope with Olympus DP 72 camera.

Quantification of positive staining was performed using ImageJ Fiji version (AEC and DAB

staining) and CellSens Standard (Olympus, glomeruli and capillary size measurement) software.

 

Isolation of mouse renal glomeruli and glomeruli permeability assay

Mouse renal glomeruli were isolated from control mice using sieving technique (3). Glomerular

permeability was measured by a determination of albumin permeability as described previously

(4) with slight modification. Briefly, 50 l of bovine serum albumin (4 g/dL BSA, isooncotic

solution, Sigma-Aldrich) solution in PBS was added into 96-well plate with or without

recombinant 1 μM MSP1 (R&D Systems) and 200 nM RONi (BMS-777607, Santa Cruz

Scientific). Glomeruli were collected by glass pipette, placed in 96-well plate (10 glomeruli per

well), incubated for 30 min at 370C in an isooncotic solution, and images were obtained for all

glomeruli. Three wells (30 glomeruli) were used for each treatment. PBS (300 μl) was added to

each well, and glomeruli were incubated at 370C for another 30 min in hypooncotic medium;

then images were obtained. Images were used for area measurement for each glomeruli by

CellSens software (Olympus) and glomerular volume was calculated. The glomerular volume

change was shown as a percent of non-treated glomeruli volume.

Immunofluorescent staining and Western Blots

Rabbit MT-SP1 (Anti-Matriptase/MT-SP1, Cat# IM1014, Calbiochem) antibody was used for

immunostaining and Western blot (WB) of THP1 cells. HGEC were treated with 1 μM of human

recombinant MSP1 (R&D Systems, Cat# 352-MS) with or without RON inhibitor (200 nM) for

15, 30, 180 minutes and 24 hrs. WB analysis was performed with rabbit anti-p44/p42 MAPK

(Erk1/2) (cat#4695), rabbit anti-phospho-p44/p42 Erk1/2 (Cat# 4377), rabbit anti-pan-Akt (Cat#

4685) and rabbit anti-phospho-Akt (Cat# 4660) antibodies (all from Cell Signaling Technology).

Mouse anti-β-actin antibodies were from Sigma-Aldrich (Cat# A-1978). Phalloidin-FITC

conjugate was from Sigma-Aldrich (Cat# P5282).

MTT assay for cell viability and growth

Cells were treated with 1 μM of MSP1 for 24 hrs. MTT solution (3-(4,5-Dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide, Sigma-Aldrich, 250 μg/ml) was added for 2 h at 370C

followed by DMSO, and optical density was read at 630 nm. Five 5 wells were analyzed for

treatment and control in three independent experiments.

Meta analysis

Analysis of gene expression profiles was performed using Geo database NCBI Data Set

GDS3203 and Data Set GDS2036.

 

Supplemental References:

1. Ryan TM, Ciavatta DJ, Townes TM. Knockout-transgenic mouse model of sickle cell

disease. Science. 1997;278(5339):873-876.

2. Wu LC, Sun CW, Ryan TM, et al. Correction of sickle cell disease by homologous

recombination in embryonic stem cells. Blood. 2006;108(4):1183-1188.

3. Misra RP. Isolation of glomeruli from mammalian kidneys by graded sieving. American

journal of clinical pathology. 1972;58(2):135-139.

4. Savin VJ, Sharma R, Lovell HB, Welling DJ. Measurement of albumin reflection

coefficient with isolated rat glomeruli. Journal of the American Society of Nephrology : JASN.

1992;3(6):1260-1269.

Supplemental Figure 1. Proliferation of glomerular cells is increased in SCDmice.(A) Representative picture of PCNA staining of renal sections (brown nuclearstaining). PCNA kit was obtained from Invitrogen (Cat#93-1143). (B)Quantification of PCNA expression in glomeruli is performed in 5 mice pergroup. Means are shown. Each dot represents a value obtained from oneglomerulus cross-section. Bar size on the microphotographs is 80 µm..

Ctrl SCD

A

B P=1.3x10-9

Ctrl SCD

A

B

Supplemental Figure 2. Renal capillary is markedly congestedin SCD mice.(A-B) Representative pictures of glomerular, cortical peritubularcapillaries (A) and papilla capillaries (B) H&E staining. Bar size is40 µm.

Supplemental Figure 3. Renal interstitial macrophage infiltration is increasedin SCD mice.(A) Representative picture of renal interstitial macrophages (F4/80)immunostaining (red color, arrows). Bar size is 120 µm. (B) Quantification ofmacrophages infiltration (5 mice per group). Means and standard deviationsare shown.

Ctrl SCD

A

B

CtrlSCD

0

300

600

900

1200

1500

Mac

rop

hag

e n

um

er, %

of

con

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l P=0.0002

Supplemental Figure 4. Low levels of iNOS expression in the infiltratingrenal macrophage in SCD mice.(A) Representative picture of macrophage immunostaining (red color,arrow). Squares show enlarged area (B). (C) Quantification of iNOSpositive macrophages per glomeruli cross section is shown. Five mice pergroup were used for each staining. Means are shown. Each dot representsa value obtained from one glomerulus cross-section. Bar sizes on themicrophotographs are 120 µm (A)) and 40 µm (B).

Ctrl SCD

A

B

CP=0.07

Ctrl+DMSO Ctrl+RONi SCD+DMSO SCD+RONi

ICA

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vWF

PA

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H&

E

Supplemental Figure 5. Treatment of SCD mice with RON inhibitor ameliorates endothelial injury.Representative picture of renal sections of control and SCD mice treated with either RONi or vehicle. (A) H&E staining. (B) PAS staining.(C) von Willebrand factor (vWF) immunostaining (red color). (C) Intercellular Adhesion Molecule (ICAM) immunostaining (red color). Bar sizes are 120 µm.

Supplemental Figure 6. Treatment of SCD mice with RON inhibitor does not reduce mesangial hyper-cellularity.Quantification of mesangial cell number per glomerular capillary loop cross-section (PAS staining). Five mice per group were used for quantification. Means and SD are shown.

P=0.0002

P=0.052